Methods for obtaining stem cells

ABSTRACT

The present invention relates to methods for obtaining stem cells from mammalian cadavers, methods for obtaining or purifying stem cells from a sample likely to contain non-stem cells, methods of regeneration of injured tissues and methods of treatment.

The present invention relates to methods for obtaining stem cells frommammalian cadavers, methods for obtaining or purifying stem cells from asample likely to contain non-stem cells, methods of regeneration ofinjured tissues and methods of treatment.

There is strong clinical and scientific interest in finding new sourcesof stem cells which are readily available, since stem cells have a greatimportance for biological studies, stem cells therapies and regenerativemedicine.

Two main types of mammalian stem cells exist: embryonic stem cells(hereinafter abbreviated “eSC”) and “adult” or “somatic” stem cells(hereinafter abbreviated “sSC”).

The former type was isolated from human embryos more than one decade ago(Thomson et al., Science, 282(5891): 1145-1147, 1998). Thomson et al.discovered a method to derive and isolate these cells from embryos andfoetal germ cells. eSC have the potential to develop into almost all ofthe more than 200 different known human body cells.

The second type of known stem cells is undifferentiated cells that arefound in differentiated somatic tissue. Generally these cells aremultipotent, i.e. having the capacity to differentiate into severaltypes of somatic cell within the tissue in which they reside.

During the last ten years, sSC have been found in many organs andtissues, including central nervous system, bone marrow, peripheralblood, blood vessels, umbilical cordon blood, skeletal muscle, epidermisof the skin, dental pulp, heart, gut, liver, pancreas, lung, adiposetissue, ovarian epithelium, retina, cornea and testis. They are thoughtto reside in a specific area of each tissue, which is called a “stemcell niche”.

In vivo, the main role of sSC is to maintain homeostasis and to replacecells that have died because of use, apoptosis, injury or disease.However, most sSC have a limited capacity to handle major trauma ordiseases that would cause a vast loss of cells and tissue.

Both types of stem cells have the ability to proliferate whilemaintaining an undifferentiated state and the capacity to give rise to asuccession of mature functional differentiated cells.

However, whereas eSC are pluripotent, i.e. they can differentiate intoalmost all cell types of the body and possess the capability ofdeveloping into any organ or tissue type, sSc are multipotent, whichmeans that sSC can only differentiate into several types of cell whichare closely related to the tissue from which sSC derive. For example, ahematopoietic stem cell may only give rise to any of the different typesof terminally differentiated blood cells.

Even if the embryonic stem cells have the greatest degree ofdifferentiation potential, they are not readily available andprocurement of these cells from embryos or foetal tissue, includingabortuses, raises religious and ethical issues.

On the contrary, several types of sSC such as mesenchymal stem cells,hematopoietic stem cells, skin stem cells, adipose-derived stromal stemcells, are more accessible and provoke less ethical controversy thaneSC. Further, in some cases stem cells can be obtained from the patientto be engrafted, i.e. the recipient which is better suitable fortransplantation purpose than eSC since autologous graft avoids the riskof rejection.

Unfortunately, sCS are rare and present in small quantity in somatictissues. Since very few stem cells are present in adult tissues,extraction procedures to recover these cells from a tissue generallyresult in contamination by other cell types such as fibroblasts.Consequently, when a tissue sample is harvested, it is necessary to sorta heterogeneous mixture comprising stem cells and non-stem cells.However, a specific cellular marker characterising one type of sSC doesnot always exist, making it difficult to isolate sSC from othercell-types of the sample. Hence, to date, isolation of normallyoccurring populations of stem cells from adult tissues is technicallydifficult and costly.

Moreover, even if sCS are more readily available than eSC, a major issuewhich remains to be solved is the acute organ and bone marrow shortagemainly due to the small number of donors, and which is increased by thedifficulty to find matching donors, especially for minority groups.

Accordingly, there is a need for a new source of readily available stemcells. Furthermore, there is also a great need for a simple method whichenables for specifically selecting any type of stem cell from biologicalmaterial comprising non-stem cells.

Recently, it has been shown that neural stem cells from rat can beisolated from the brain of deceased adult or early postnatal rats even 6days after death when rats were stored at 4° C. (Yi Xu et al., Journalof Neuroscience Research, 74: 533-540, 2003). Then, Yi Xu et al.suggested using cadavers as a new source of neural stem cells usable forclinical purposes. Further, the study of Yi Xu et al. showed that theamount of stem cells obtained from rat cadavers depends on the age ofthe rats, since there are significantly more neural stem cells in theearly postnatal rats than in the adults. In addition, Yi Xu et al.showed that from day 2 post-mortem the number of neural stem cellsstrongly decreases and that only few neural stem cells survive 4 daysafter death in 4 weeks old rats.

A recent study performed on human neural stem cells indicates that thehighest proliferation rate is obtained when these cells are cultured inhypoxic conditions, at an oxygen concentration between 2.5 and 5%, while1% of oxygen is detrimental for cell survival (Santilli et al., PLOSOne, 5(1): e8575, 2010). This study suggests that an oxygenconcentration of less that 2.5% is noxious for stem cells.

Another study conducted on human abortuses preserved at 4° C. clearlyshowed that the number of viable neural stem cells decreased sharplywhen preservation is prolonged to 12 hours after death (Xinchun Liu andal., Journal of Neuroscience methods, 157: 64-70, 2006). Viable neuralprogenitor cells from human cadavers were also obtained from braintissue 20 hours after death (Palmer et al., Nature, 411: 42-43, 2001).

It was also proposed in the International Application WO 01/53462 to usetissues of cadavers as a source of progenitors and stem cells, inparticular as a source of progenitor cells having the capacity todevelop into hepatocytes and biliary cells. WO 01/53462 recommended touse tissues which had been harvested within about six hours after thedonor's heartbeat ceased, and a maximum of 30 hours post-mortem wasindicated for liver tissue. However, it is to be noted that thisapplication showed experiments only for liver progenitor cells whichcome from livers obtained not later than 30 hours after death.

It was also shown that the cell viability of adult rat neural stem cellswhen cultured 24 hours in anoxic condition at 37° C. increased up to 60%compared to normoxic condition (Bürgers et al., Exp. Brain Res., 188(1):33-43, 2008), and that cell division activity increased from 2% innormoxic condition to 16% in anoxic condition. However, the effect oflonger periods in anoxia on viability of stem cells was not tested andthe transplantation potential of stem cells was not assessed.

Concerning neural stem cells, histological and immunohistochemicalassays suggest that the rich vascular bed presents in brain, inparticular the subventricular zone, might be an important elementresponsible for survival of neural stem cells during the post-mortemperiod and can likely act as a niche for the maintenance of neural stemcells (Yi Xu et al., Journal of Neuroscience Research, 74: 533-540,2003).

As indicated above, the prior art mainly concerns neural stem cells anddoes not demonstrate that other type of stem cells can be obtained fromcadavers, except liver stem cells. Further, the longest period of timefollowing death after which viable stem cells could be found was 6 daysfor rat neural stem cells. Viability of human neural stem cells wasassessed only until 20 hours post mortem.

In addition, because the biological environment of neural stem cells isspecific and not found in other tissues, data of the prior art relatingto neural stem cells do not allow to conclude that other types of stemcells can be obtained from cadavers.

Unexpectedly, the inventors have observed that stem cells, in particularhuman skeletal muscle stem cells and hematopoietic stem cells, have thecapacity to resist in the absence of oxygen for a period of time longerthan the periods of time previously reported in the art. In particular,the inventors have shown that stem cells from human cadavers can resistfor up to 17 days after death, and resist better than non-stem cells todeprivation of oxygen.

They also shown, firstly that mouse skeletal muscle stem cells cansurvive at least 10 days after death and can reform muscle in vitro, andsecondly that bone marrow harvested at least until 4 days after deathcan be efficiently transplanted and eventually serially transplanted inirradiated mice to reconstitute bone marrow, demonstrating that the stemcells even long term repopulating stem cells are viable and functionalin vivo.

These results indicate that cadavers, even several days after death, arean important source of stem cells, in particular of muscle stem cells(especially muscle stem cells obtained from skeletal muscle or smoothmuscle) and hematopoietic stem cells, useful for stem cells therapiesand regenerative medicine.

Method for Obtaining Stem Cells from a Cadaver

Accordingly, the present invention is directed to a method for obtainingmammalian stem cells which comprises the following steps:

a) harvesting a tissue from a mammalian cadaver stored at 1-6° C., theharvesting being performed on a part of the cadaver's body in which stemcells are usually present in a living counterpart; and

b) extracting mononuclear cells from the harvested tissue.

The mammalian cadaver has been stored at 1-6° C., preferably at 3-5° C.,and ideally at 4° C. as soon as possible after the death, preferablyfrom 0 minutes to 48 hours after death, and in order of preference from0 minutes to 24 hours, from 0 minutes to 12 hours and from 0 minutes to6 hours after death.

More preferably, the cadaver has been stored at 4° C. within 24 hoursafter death.

The cadaver may be an embryo, a foetus, a neonate, an infant, a child, ajuvenile or an adult.

The mammalian cadaver may be a human or a non-human cadaver, e.g.rodent, canine, feline, primate, equine, ovine, bovine, caprine species.According to preferred embodiment of the invention, the mammaliancadaver is a human cadaver.

Tissue harvested up to 30 days after death can be efficiently used toperformed the method according to the present invention.

In particular, the tissue harvesting step a) may be performed after aperiod of time following death comprised between:

-   -   0 minute and 30 days, 0 minute and 25 days, 0 minute and 20        days, 0 minute and 15 days, 0 minute and 10 days, or 0 minute        and 5 days;    -   preferably between 20 hours and 30 days, 20 hours and 25 days,        20 hours and 20 days, 20 hours and 15 days, 20 hours and 10        days, or 20 hours and 5 days;    -   still preferably between 1 day and 30 days, 1 day and 25 days, 1        day and days, 1 day and 15 days, 1 day and 10 days, or 1 day and        5 days;    -   still preferably between 2 days and 30 days, 2 days and 25 days,        2 days and 20 days, 2 days and 15 days, 2 days and 10 days, or 2        days and 5 days;    -   still preferably between 3 days and 30 days, 3 days and 25 days,        3 days and 20 days, 3 days and 15 days, 3 days and 10 days, or 3        days and 5 days.

In a preferred embodiment, the temperature of the mammalian cadaver is1-6° C. before the tissue harvesting step a) is performed.

The longest period of time after which the tissue could not be harvestedbecause it would not contain viable stem cells any more (in part due totissue decomposition) depends on the mammalian specie and the type ofstem cells, and can be easily determined by one of ordinary skill in theart. For instance, for human muscular stem cells, this period of time iscomprised between 20 and 30 days.

In the present invention, “death” is intended to mean the moment whenthe heart of the mammalian definitively stops beating.

The tissue harvested at step a) comes from a part of cadaver's body inwhich stem cells are usually present in a living counterpart, includingcentral nervous system (brain and spinal cord), bone marrow, peripheralblood, blood vessels, umbilical cord blood, muscle (in particularskeletal muscle or smooth muscle), epidermis of the skin, dental pulp,heart, gut, liver, pancreas, lung, adipose tissue, ovarian epithelium,retina, cornea and testis.

Once the tissue harvested from cadaver, it may be used immediately instep b), or it may be stored before being used in step b) at 1-10° C.,preferably at 1-6° C., more preferably at 3-5° C. and still morepreferably at 4° C., in normoxia conditions (oxygen concentration ofabout 21%), preferably in hypoxia conditions (oxygen concentration ofless than 5%), and more preferably at an oxygen concentration equal toor of less than 0.1%, preferably in the absence of oxygen. In any case,the harvested tissue should not be stored longer than the maximum periodof time after which the tissue does not contain viable stem cells anymore. As indicated above, this time period depends on the mammalianspecies.

The term “extracting mononuclear cells”, as use herein, refers to invitro separation of mononuclear cells from other type of cells presentin the harvested tissue. Standard methods to carry out extraction ofmononuclear cells are well known by one skilled in the art. For example,one can use the following methods: mechanical dissociation of the tissue(i.e. the tissue is minced into small pieces) followed by enzymaticdigestion (using proteolytic enzymes such as pronase, trypsin, dispase,collagenase and/or an association of several enzymes (Chazaud et al.,Exp. Cell. Res., 258(2): 237-44, 2000)) and/or or non-enzymaticprocedures (Ca2+ chelation) and/or an association of mechanicaldissociation and enzymatic digestion combined to immunomagnetic cellsorting or FACS-cell sorting (Arnold L, et al., J. Exp. Med.,204(5):1057-69, 2007) and/or association of mechanical dissociation,enzymatic or non-enzymatic digestion followed by Fluorescence-ActivatedCell Sorting (FACS) after multiple immunostainings using appropriatemarkers (for human muscle stem cells CD56+ CD45−). When cells are insuspension such as in blood or in bone marrow, one can use Ficoll Paqueplus (Amersham Biosciences) density gradient or adhesion steps(Friedenstein A J, Calci Tissue Int, 56:S17, 1995), as well asimmunomagnetic or FACS cell sorting (Prospective isolation ofmesenchymal stem cells from multiple mammalian species usingcross-reacting anti human monoclonal antibodies).

Antibiotics and antifungus (such as an association of penicillin,streptomycin, gentamycin and amphotericin B) are used to preventbacterial/fungal contamination of the cell preparation.

The “term mononuclear cell” is intended to mean any cell i) which has anot lobated nucleus, nor multinucleated nucleus, or ii) which has asingle nucleus and is preferably able to be dissociated from the tissueand collected in suspension.

In a preferred embodiment, the method comprises an additional step b′)following step b) consisting of culturing the extracted mononuclearcells.

Typically, in step b′) the extracted mononuclear are cultured for aperiod of time of 1 day to 30 days, 1 day to 25 days, 1 day to 20 days,1 day to 15 days, 1 day to 10 days, or 1 day to 5 days, preferablybetween 2 days to 30 days, 2 days to 25 days, 2 days to 20 days, 2 daysto 15 days, 2 days to 10 days, or 2 days to days, still preferablybetween 3 days to 30 days, 3 days to 25 days, 3 days to days, 3 days to15 days, 3 days to 10 days, or 3 days to 5 days. For mononuclearextracted from human tissue, culturing step c) is performed preferablyfor a period of time of 5 to 20 days and more preferably from 7 to 14days.

Step b′) consisting of culturing the mononuclear cells extracted in stepb) is conducted at 1-37° C., preferably at 20-37° C., still preferablyat 25-37° C., still preferably at 37° C., and at an oxygen concentrationequal to or less than 21% and preferably at an oxygen concentrationequal to or less than 0.1% to 10%, still preferably from 0 to 5%, stillpreferably from 0 to 3.5% and more preferably at 3.5%. Preferably, stepc) is conducted at a CO₂ concentration of 5%.

Culture medium used to perform step b′) can be any suitable chemicallydefined culture medium commonly used for culturing mammalian cells. Sucha culture medium is well known to those of ordinary skill in the art.For instance it can be MEM, Dulbecco's Modified Eagle's medium, ormodified MEM or DMEM, RPMI 1640 media, CMRL-1066 Medium, Ames' Medium,Ham's F10 and F12 media, Leibovitz's, Williams media

Preferably, the culture medium is supplemented with at least one of thegrowth factors usually used for culturing mammalian cells and which arewell known from the skilled person in the art. Suitable growth factorscan be selected from the group comprising Fibroblast growth Factors (forinstance FGF-1 and FGF-2), Erythropoietin or others colony stimulatingfactors Epidermal GF (EGF), Insulin (IGF), Stromal GF, Interleukin-1,-3, -6, Flt3-ligand. Growth factors will be selected according to thetype of stem cell to be cultured.

Further, feeder cells can be used in step b′) to provide nutrients tostem cells and to maintain stem cells in an undifferentiated state.Feeder cells suitable for the cultivation of stem cells are well knownfrom the one skilled in the art and can be chosen from the groupcomprising the mouse embryonic fibroblasts and human embryonicfibroblasts.

According to an advantageous embodiment of the invention, the culturemedium is supplemented with glucose at a final concentration from about0.5 g/liter to 4.5 g/liter and/or with human or foetal calf serum (orbovine serum substitute) at a final concentration comprised betweenabout 2.5 and 20%. By “about”, it is meant that a slightly lower orhigher quantity of glucose or serum can be used. When the stem cellsobtained by the method subject of the present invention are intended tobe introduced in human body, for example in a graft purpose, human orfoetal calf serum is replaced by a chemically defined composition (e.i.serum substitute) which acts as sera. Such chemically definedcompositions are well known by a person having ordinary skill in theart, and are for example ultroserG™ or platelet lysate (see Bernardo ME, J. Cell. Physiol., 207; 211: 121-130).

In a particular embodiment of the method, step a) is performed ontrabecular bone or peripheral blood, the harvested tissue is bone marrowor peripheral blood and the stems cells obtained by the method arehematopoietic stem cells or mesenchymal stem cells.

Peripheral blood is intended to mean circulating blood in a otherwiseliving mammal, i.e. the cellular components of blood which are found inthe vessels or in the arteries and which is not sequestered within thelymphatic system, spleen, liver, or bone marrow.

In another particular embodiment of the method, step a) is performed onmuscle, especially skeletal muscle or smooth muscle, the harvestedtissue is muscle sample, especially skeletal muscle sample or smoothmuscle sample, and the stems cells obtained by the method are musclestem cells.

In the context of the invention, the term muscular stem cells isintended to mean satellite cells, myogenic precursor cells interstitialmuscle or mesenchymal stem cells, preferably obtained from skeletalmuscle sample or smooth muscle sample.

In still another particular embodiment of the method, step a) isperformed on a nervous tissue such as brain or spinal cord or meninges,preferably at least 24 hours after death, the harvested tissue beingbrain or spinal cord sample and the stems cells obtained by the methodbeing neural stem cells.

The method according to the invention is suitable for obtaining anytypes of stem cells, for instance embryonic or foetal stem cells, otheradult stem cells such as epithelium stem cells (skin, digestive tract,respiratory tract, oral mucosa, genital mucosa), germ cells, eye stemcells, cancer stem cells.

When the stem cells obtained from a cadaver are intended to beadministered to a subject in need thereof, for instance for regeneratingan injured tissue, for treating acquired, congenital or geneticdisorders (e.g. muscle or neural disorders), for treating malfunction ordisease (e.g. hematopoietic system malfunction or disease), the methodpreferably comprises an ultimate step following step b), or step b′)where appropriate, consisting of resuspending the extracted orcultivated mononuclear cells in a pharmaceutically acceptable carrier.The cell suspension thus obtained is suitable for being administered toa subject.

The pharmaceutically acceptable carrier used in this ultimate stepshould neither be prejudicial for stem cells viability and functions,nor be toxic for a subject in need to be administered with thecomposition.

Non-limiting examples of pharmaceutically acceptable carriers includesaline solution, i.e. a solution having the same osmolarity as blood(e.g. a solution of 0.90% w/v of NaCl, about 300 mOsm/L), Ringer'ssolution, lactated Ringer's solution, or acetated Ringer's solution.

The obtained mammalian stem cells from cadaver may also be used forpreparing transgenic mammalian stem cells expressing a polynucleotidesequence of interest. The invention thus also relates to a method forobtaining mammalian stem cells expressing a transgene of interest,wherein the mononuclear cells extracted from the harvested tissue ofstep b), or the cultured mononuclear cells of step b′) whereappropriate, are transformed or transfected with a vector, especially avector of expression, or transduced with a virus vector, preferably aretrovirus vector, advantageously a lentivirus vector, comprising atleast one polynucleotide sequence of interest, so that said at least onepolynucleotide sequence of interest is expressed by the mononuclearcells.

In the context of the invention, the term “to transform” means theintroduction of a “foreign” (i.e. extrinsic or extracellular)polynucleotide sequence (i.e. gene, portion of a gene, DNA or RNAsequence) of interest into a host cell, so that the host cell willexpress the introduced gene, portion of a gene, or sequence to produce adesired substance, typically a protein or enzyme encoded by theintroduced gene or sequence of interest. The term “to transfect” meansthe introduction of a foreign nucleic acid into a cell. The term “totransduce with a retrovirus vector” is intended to mean the stableintroduction of a “foreign” polynucleotide sequence of interest into thegenome of a stem cell by infecting said stem cell with an infectiousretrovirus vector whose genome sequence comprises the polynucleotidesequence of interest.

In the context of the invention, the term “Polynucleotide sequence ofinterest” (also called “transgene”) means any gene, portion of a gene,DNA or RNA sequence that is desired to be expressed in the mononuclearcell of step b), or step b′) where appropriate. A polynucleotidesequence of interest can be homologous or heterologous to themononuclear cell genome.

Methods for engineering vectors (viral and non-viral vectors),especially expression vectors, and for transforming, transfecting ortransducing mammalian stem cells with a vector, especially a vector ofexpression, comprising at least one polynucleotide sequence of interestso that said cells express a polynucleotide sequence of interest (calledtransgenic stem cells) are well known in the art, and are for instancedescribed by Cossu and colleagues (Dellavalle A, Sampaolesi M,Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L, Innocenzi A, Galvez BG, Messina G, Morosetti R, Li S, Belicchi M, Peretti G, Wright W E,Torrente Y, Ferrari S, Bianco P, Cossu G Pericytes of human skeletalmuscle are myogenic precursors distinct from satellite cells. Nat CellBiol. 2007 March; 9(3):255-67. Epub 2007 Feb. 11) or Chamberlain (Li S,Kimura E, Ng R, Fall B M, Meuse L, Reyes M, Faulkner J A, Chamberlain JS. A highly functional mini-dystrophin/GFP fusion gene for cell and genetherapy studies of Duchenne muscular dystrophy. Hum Mol. Genet. 2006 May15; 15(10):1610-22. Epub 2006 Apr. 4).

When the transgenic stem cells obtained from a cadaver according to themethod above recited are intended to be administered to a subject inneed thereof, the method preferably comprises an additional stepconsisting of resuspending the transgenic stem cells in apharmaceutically acceptable carrier to obtain a composition suitable forbeing administered to a subject.

The transgene may be any polynucleotide sequence useful for treating adisease.

For instance, when stem cells are muscle stem cells, the transgene maybe a polynucleotide sequence encoding dystrophin, calpain, lamin,dysferlin, caveolin, sarcoglycan, myotilin, nemaline, desmin, enzymessuch as mitochondrial enzymes, or glycolytic enzymes or growth factors.

Selection of Stem Cells by Exposure to Anoxia

Furthermore, the inventors unexpectedly found that culturing abiological sample comprising stem cells and other cell types in theabsence of oxygen triggers death of non-stem cells whereas stem cellssurvive. Since resistance to anoxia seems to be a general property ofstem cells, and because non-stem cells and the great majority of cancercells do not resist after few hours or few days of anoxia, the inventorstook advantage of this property for the enrichment and purification of abiological sample for stem cells.

Consequently, it is another object of the present invention to provide asimple method which leads to the depletion of non-stem cells, such ascancer cells, which may be present and contaminate a harvested tissue,and promotes the enrichment and the purification of stem cells.

In the context of the invention, the term “non-stem cell” is intended tomean any type of cell which is not able to proliferate while maintainingan undifferentiated state and which does not have the capacity to giverise to a succession of mature functional differentiated cells.

The term “cancer cell” is intended to mean a cell which displaysuncontrolled growth and which has the capacity to invade adjacenttissues in vivo. This term encompasses cancer cells derived from stemcells.

Accordingly, the invention is also directed to a method for obtainingstem cells comprising the following step:

a) maintaining biological material which usually comprises stem cells atan oxygen concentration equal to or less than 0.1%;

b) selecting viable cells, wherein viable cells are stem cells.

In step a), the biological material is maintained in these conditionsduring a period of time sufficient to trigger apoptosis and death ofnon-stem cells without affecting viability of most of the stem cellscomprised in the biological material.

According to preferred embodiment of the invention, the biologicalmaterial is submitted to mechanical dissociation of the tissue (i.e. thetissue is minced into small pieces) followed by enzymatic digestion(using pronase, trypsin, dispase, and/or collagenase or associationbefore carrying out step a) and/or non-enzymatic procedures (Ca2+chelation) and/or magnetic cell sorting and/or Fluorescence-ActivatedCell Sorting (FACS). The period of time necessary to kill non-stem cellswithout killing stem cells will easily be determined by a person havingordinary skill in the art and will be adapted according to the type ofnon-stem cells and stem cells present in the biological sample to betreated.

Typically, this period of time is comprised between 12 hours and 60days,

-   -   preferably between 1 day and 30 days, 1 day and 25 days, 1 day        and 20 days, 1 day and 15 days, 1 day and 10 days, or 1 day and        5 days;    -   still preferably between 2 days and 30 days, 2 days and 25 days,        2 days and 20 days, 2 days and 15 days, 2 days and 10 days, or 2        days and 5 days;    -   still preferably between 3 days and 30 days, 3 days and 25 days,        3 days and 20 days, 3 days and 15 days, 3 days and 10 days, or 3        days and 5 days;    -   still preferably between 4 days and 30 days, 4 days and 25 days,        4 days and 20 days, 4 days and 15 days, 4 days and 10 days, or 4        days and 5 days;    -   still preferably between 5 days and 30 days, 5 days and 25 days,        5 days and 20 days, 5 days and 15 days, 5 days and 10 days.

Typically, the temperature at which step a) may be performed iscomprised between 1-37° C., and in order of preference 1-6° C., 3-5° C.,and 4° C.

Preferably, the biological material is maintained at 1-37° C. and at aconcentration of less than 0.1% of oxygen for 2 to 20 days.

Step b) is intended to purify stem cells by removing most of dead cellsor cell debris from stem cells. This step may be achieved for instanceby sorting living stem cells using Fluorescence-Activated Cell Sorting(FACS) and a marker which is specifically expressed by living cells, forinstance calcein or by exclusion of dead cells positive for annexin V orpropidium iodide, or by using physical separation procedures such asdifferential centrifugation.

In step a), the biological material is maintained in a culture mediumwhich may be MEM, Dulbecco's Modified Eagle's medium, or modified MEM orDMEM, RPMI 1640 media, CMRL-1066 Medium, Ames' Medium, Ham's F10 and F12media, Leibovitz's, Williams media.

According to a preferred embodiment of the invention, the method isperformed in the absence of oxygen.

In another preferred embodiment of the method, biological material is acell culture, including cell culture derived from brain, spinal cord,bone marrow, peripheral blood, blood vessels, umbilical cordon blood,muscle (especially skeletal muscle or smooth muscle), epidermis of theskin, dental pulp, heart, gut, liver, pancreas, lung, adipose tissue,ovarian epithelium, retina, cornea and testis.

Preferably, the biological material is a bone marrow or muscular cellculture, especially skeletal muscular cell culture or smooth muscularcell culture.

In a preferred embodiment of the method the biological material isselected from the group consisting of bone marrow and circulating orperipheral blood, and the obtained stem cells are hematopoietic stemcells, peripheral or circulating hematopoietic stem cells or mesenchymalstem cells.

In still a preferred embodiment of the method the biological material ismuscle, especially skeletal muscle sample or smooth muscle sample, andthe obtained stem cells are muscular stem cells.

In another preferred embodiment of the method the biological material isselected from the group comprising brain, spinal cord and meningessample, and wherein the enriched stem cells are neural stem cells.

The stem cells obtained by the method of the invention are suitable forbeing transplanted into a recipient in need thereof.

The biological sample can be obtained from a living donor, except humanembryo or foetus, or from a cadaver stored at 1-6° C., preferably at3-5° C., still preferably at 4° C., directly after death, preferablyfrom 0 minutes to 48 hours after death, and in order of preference from0 minutes to 24 hours, from 0 minutes to 12 hours and from 0 minutes to6 hours after death.

The biological sample can be obtained from a human or a non-human donor,e.g. rodent, canine, feline, primate, equine, ovine, bovine, caprinespecies. According to preferred embodiment of the invention, themammalian is a human.

The donor may be living or not.

When the stem cells obtained after culture in anoxia (oxygenconcentration equal to or less than 0.1%) are intended to beadministered to a subject in need thereof, for instance for regeneratingan injured tissue, for treating acquired, congenital or geneticdisorders (e.g. muscle or neural disorders), for treating malfunction ordisease (e.g. hematopoietic system malfunction or disease), the methodpreferably comprises an ultimate step c) following step b) consisting ofresuspending the viable cells of step b) in a pharmaceuticallyacceptable carrier to obtain a composition suitable for beingadministered to a subject.

The pharmaceutically acceptable carrier used in this ultimate stepshould neither be prejudicial for stem cells viability and functions,nor be toxic for a subject in need to be administered with thecomposition.

Non-limiting examples of pharmaceutically acceptable carriers includesaline solution, i.e. a solution having the same osmolarity as blood(e.g. a solution of 0.90% w/v of NaCl, about 300 mOsm/L), Ringer'ssolution, lactated Ringer's solution, or acetated Ringer's solution.

The method for obtaining mammalian stem cells by exposure to anoxia mayalso be used for obtaining transgenic mammalian stem cells expressing apolynucleotide sequence of interest (also called “ransgene”). In thismethod for obtaining mammalian stem cells expressing a gene, or portionof a gene, of interest, the viable cells selected in step b) aretransformed, transfected or transduced with a vector, especially avector of expression, so that at least one polynucleotide sequence ofinterest is expressed by the mononuclear cells.

When the transgenic stem cells obtained by exposure to anoxia areintended to be administered to a subject in need thereof, the methodpreferably comprises an additional step consisting of resuspending thetransgenic stem cells in a pharmaceutically acceptable carrier to obtaina composition suitable for being administered to a subject.

The transgene may be, for example, any polynucleotide sequence usefulfor treating a disease.

For instance, when stem cells are muscle stem cells, the transgene maybe a polynucleotide sequence encoding the dystrophin, calpain, lamin,dysferlin, caveolin, sarcoglycan, myotilin, nemaline, desmin, enzymessuch as mitochondrial enzymes, or glycolytic enzymes or growth factors.

Isolated Muscular Stem Cells

The inventors have found that the expression level of some markers ofmyogenic cell commitment and of stem-like state are specific of the postmortem derived muscle stem cells.

Therefore, another object of the invention relates to an isolated musclestem cell characterized in that it does not express myogenin gene(Myogenin⁻).

Preferably, the isolated muscle stem cell of the invention isPax7^(high) and/or of CD34⁺. The Pax7^(high) level may be determined asa level of expression of Pax7 significantly higher than the level ofPax7 in muscle stem cells isolated from a living subject and in aresting tissue.

Preferably also, the isolated muscle stem cell of the invention has alevel of mitochondrial activity (which may be determined by measuringthe number of active mitochondria) lower than muscle stem cells isolatedfrom living subject and in a resting tissue).

In an embodiment, the isolated muscle stem cell of the invention is atransgenic cell which expresses a “foreign” polynucleotide of interest(also called “transgene”), as described above.

The transgene may be, for example, any polynucleotide sequence usefulfor treating a disease or a disorder of muscles.

For instance, when stem cells are muscle stem cells, the transgene maybe a polynucleotide sequence encoding dystrophin, calpain, lamin,dysferlin, caveolin, sarcoglycan, myotilin, nemaline, desmin, enzymessuch as mitochondrial enzymes, or glycolytic enzymes or growth factors.

In another embodiment, the isolated muscle stem cell or the isolatedtransgenic muscle stem cell are formulated in a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluent.Said carrier and diluent should not be prejudicial for stem cellsviability and functions and should not be toxic for a subject in need tobe administered with the composition. Suitable examples of carriers anddiluents have been described above.

Therapeutic Applications

Again another object of the invention relates to method for regeneratingan injured tissue, wherein stem cells suitable for regenerating theinjured tissue are obtained by any of the methods of the inventiondisclosed above, and wherein the stem cells are introduced into thebody, preferably into the injured tissue, of a patient in need thereof.

The present invention also relates to stem cells obtained or selected bya method of the invention for obtaining stem cells as described above,for use in the regeneration of an injured tissue.

The stem cells may have been transformed to express a transgene.

The stem cells may be introduced into the body parenterally via avascular vessel (small or large arteries, small or large veins), orintroduced directly into the organ comprising the injured tissue.

The injured tissue to be regenerated by the method can be any tissue ofthe body, including brain, spinal cord, bone marrow, peripheral blood,blood vessels, umbilical cordon blood, muscle (especially skeletalmuscle or smooth muscle), epidermis of the skin, dental pulp, heart,gut, liver, pancreas, lung, adipose tissue, ovarian epithelium, retina,cornea and testis.

Preferably, the injured tissue to be regenerated is selected from thegroup consisting of bone marrow, muscle (especially skeletal muscle orsmooth muscle), brain and spinal cord.

When the injured tissue is brain or spinal cord, the stem cells suitablefor regenerating the injured tissue are neural stem cells obtained by amethod for obtaining stem cells disclosed above.

When the injured tissue is blood tissue or bone marrow, the stem cellssuitable for regenerating the injured tissue are hematopoietic stemcells obtained by a method for obtaining stem cells disclosed above.

When the injured tissue is muscle tissue, especially skeletal muscletissue or smooth muscle tissue, the stem cells suitable for regeneratingthe injured tissue are muscle stem cells obtained by methods forobtaining stem cells disclosed above. In particular, muscle stem cellsmay be the Myogenin⁻ stem cells, and preferably Pax7^(high) CD34⁺,according to the invention.

The invention also relates to a method for treating hematopoietic systemmalfunction or disease, wherein hematopoietic stem cells obtained by anyof the methods of the invention for obtaining or selecting stem cells asrecited above are introduced into the body of a patient in need thereof.Preferably, the hematopoietic stem cells are introduced into thevascular system of the patient.

Another object of the invention relates to hematopoietic stem cellsobtained or selected by a method as discloses above, for use in thetreatment of hematopoietic system malfunction or disease.

The hematopoietic system malfunction or disease to be treated by themethod may be selected from the group comprising:

-   -   Leukemias and lymphomas, including Acute myelogenous leukemia,        Acute lymphoblastic leukemia, Chronic myelogenous leukemia,        Chronic lymphocytic leukemia, Juvenile myelomonocytic leukemia,        Hodgkin's lymphoma, Non-Hodgkin's lymphoma;    -   Multiple myeloma and other plasma cell disorders;    -   Severe aplastic anemia and other marrow failure states,        including Severe aplastic anemia, Fanconi anemia, Paroxysmal        nocturnal hemoglobinuria (PNH), Pure red cell aplasia,        Amegakaryocytosis/congenital thrombocytopenia;    -   SCID and other inherited immune system disorders, including        Severe combined immunodeficiency (SCID, all sub-types),        Wiskott-Aldrich syndrome;    -   Hemoglobinopathies, including Beta thalassemia major, Sickle        cell disease;    -   Hurler's syndrome and other inherited metabolic disorders,        including Hurler's syndrome (MPS-IH), Adrenoleukodystrophy,        Metachromatic leukodystrophy;    -   Myelodysplastic and myeloproliferative disorders, including        Refractory anemia (all types), Chronic myelomonocytic leukemia,        Agnogenic myeloid metaplasia (myelofibrosis);    -   Familial erythrophagocytic lymphohistiocytosis and other        histiocytic disorders.

The invention also relates to method for treating acquired, congenitalor genetic muscle disorders, trauma, such as crush or radiation injuriesof muscles, or urethral or anal sphincter incompetence (incontinence),or myocardial infarcts, or to increase the muscle mass, for instance fortreating a loss of muscle mass in older persons or in persons lain downfor a long period of time, wherein muscle stem cells obtained by any ofthe methods of the invention for obtaining or selecting stem cells asrecited above are introduced into the body of a patient in need thereof.Preferably, the muscle stem cells are introduced directly into muscle ofthe patient in need thereof. In another preferred embodiment, the musclestem cells are administrated into an artery to obtain a systemicdistribution of the stem cells.

The invention also relates to muscle stem cells obtained or selected bya method of the invention as defined above, for use in the treatment ofacquired, congenital or genetic muscle disorders, trauma, such as crushor radiation injuries of muscles, or urethral or anal sphincterincompetence (incontinence) or myocardial infarcts.

The acquired, congenital or genetic muscle disorder to be treated isselected from the group comprising:

A) Genetically determined myopathies including

(i) X-linked muscular dystrophies (Duchenne and Becker);

(ii) autosomal dystrophies including limb-Girdle muscular dystrophies(such as Miyoshi or distal myopathy, dysferlinopathies,caveolinopathies, sarcoglycanopathies, myotilinopathies), congenitalmuscular dystrophies, fascio-scapulo-humeral myopathy andoculo-pharyngeal dystrophy;

(iii) Myotonic dystrophies and non dystrophic myotonias hereditary ornot (DM1 or Steinert, DM2 or proximal myotonic myopathies);

B) Congenital myopathies without or with structural anomalies (nemalinemyopathy, myo-tubular or centro-nuclear myopathies, central-core orother core disease), for instance Desmin myopathies;C) Metabolic myopathies including

(i) Mitochondrial myopathies (such as MELAS, MNGIE, MERRF, . . . );

(ii) Lipid myopathies;

(iii) Glycogenosis (such as Mc Ardle disease, pompe disease, Taruidisease).

The invention also relates to a method for treating neural disorders,wherein neural stem cells obtained by any of the methods of theinvention for obtaining stem cells as described above are introducedinto the body of a patient in need thereof.

The invention also relates to neural stem cells obtained by a method ofthe invention as disclosed above, for use in the treatment of neuraldisorders.

In a particular embodiment, the neural disorder to be treated isselected from the group comprising;

A) Neurodegenerative disorders characterized with neuronal loss such as:

-   -   i) dementia or cortical degeneration including Alzheimer's        disease, fronto-temporal lobar atrophy, dementia with Lewy        Bodies, Pick disease, dementia linked to chromosome 17;    -   ii) Movement disorders including Parkinson disease, Progressive        supra-nuclear palsy, multiple systeme atrophy, cortico-basal        degeneration, Huntington disease, distonias, cerebellar ataxia,        hereditary spastic paraparesis;        B) Primary disease of the white matter such as:    -   i) Leukodystrophies; and    -   ii) white matter disease with inflammation such as multiple        sclerosis;        C) Ischemic vascular pathology.

The stem cells used in the above recited method of regeneration of aninjured tissue and method of treatment are provided in apharmaceutically acceptable carrier or diluent which is not prejudicialfor stem cells viability and functions not toxic for a subject in needto be administered with the composition. Suitable examples of carriersand diluents have been described above.

Further, the amount of stem cells used in the above recited method ofregeneration of an injured tissue and method of treatment is atherapeutically effective amount. A therapeutically effective amount ofstem cells is that amount sufficient to achieve tissue repair orregeneration, or to treat or contribute to the treatment of a givendisease, malfunction or condition without causing overly negativeeffects in the subject to which the stem cells are administered. Theexact amount of stem cells to be used and the composition to beadministered will vary according to the age and the weight of thepatient being treated, the type of injury, malfunction or decease, themode of administration, the frequency of administration as well as theother ingredients in the composition which comprises the stem cells.

Preferably, at least about 1.0×10⁵ cells/kg, at least about 5.0×10⁵, atleast about 1.0×10⁶, at least about 5.0×10⁶, at least about 1.0×10⁷, atleast about 5.0×10⁷, at least about 1.0×10⁸, at least about 5.0×10⁸, orat least about 1.0×10⁹ cells/kg is used for any treatment. These amountsof stem cells can be delivered to the patient in need thereof in onetime or in a sequential way.

Further, it is to be noted that when the disease or the disorders to betreated is not a genetic condition, the administered stem cells can besourced from the patient (autograft). On the contrary, when the diseaseor the disorders to be treated is a genetic condition, the patient to betreated can not be the stem cells donor (allograft).

Stem Cell Culture System

The present invention also relates to the use of an anaerobic cellculture system for culturing mammalian stem cells at an oxygenconcentration equal to or less than 0.1%, preferably less than 0.1%, andmore preferably in the absence of oxygen.

Any known device usually intended to grow anaerobic bacteria is suitablefor culturing mammalian. It is preferably totally sterile, i.e. free ofany bacteria. Such a device can be a sealed container which achieves ananaerobic environment by chemical reaction, for instance by using astarting chemical material present into the device which reacts withoxygen to deprive the oxygen which is originally contained in thecontainer. It is well understood that the starting chemical productssupplied is preferably sterile and must not be toxic for stem cells, andthat reaction products must not have detrimental effect on stem cellsviability or functions. One can also use a device such as an incubatorable to inject N2 instead of oxygen since such a device can alsomodulate the level of O2 for culture.

Such suitable devices and chemical products are well known by those ofordinary skill in the art, and include Genbag®, Genbox® (biomerieuxclinical diagnostic), Gaspak® (BD diagnostic systems), Anaerocult® (vwrinternational). According to a preferred embodiment of the invention,mammalian stem cells are cultured at 1-6° C., preferably 3-5° C., stillpreferably at 4° C. for 12 hours to 30 days.

Another object of the invention relates to the use of an anaerobic cellculture system for obtaining (selecting for) stem cells by maintainingin anoxia, i.e. at an oxygen concentration equal to or less than 0.1%,preferably less than 0.1%, and more preferably in the absence of oxygen,a biological sample comprising stem cells and non-stem cells.

The mammalian stem cells may be maintained at 1-37° C. for 2 hours to 30days 6 hours to 30 days, preferably 12 hours to 30 days and morepreferably 24 hours to 30 days.

According to a preferred embodiment of the invention, mammalian stemcells are maintained at 1-6° C., preferably 3-5° C., still preferably at4° C. for 2 hours to 30 days, 6 hours to 30 days, preferably 12 hours to30 days and more preferably 24 hours to 30 days.

The present invention will be further illustrated by the additionaldescription and drawings which follow, which refer to examplesillustrating the obtaining of stem cells from cadavers, the enrichmentof stem cells by maintaining them at 4° C. in the absence of oxygen andthe use of hematopoietic stem cells and muscle stem cells obtained bythis method of enrichment for regenerating bone marrow of irradiatedmice or skeletal muscle of mice. It should be understood however thatthese examples are given only by way of illustration of the inventionand do not constitute in anyway a limitation thereof.

FIG. 1.A. illustrates the percentage of animals giving rise to myogeniccultures (after 14 days in culture) every 4 days after death. N=10 ateach time point.

FIG. 2.A. illustrates the number of viable stem cells extracted from onemg of muscle of mouse cadaver up to 8 days after death. The number ofviable cells was evaluated by Flow Cytometry on calcein incorporationand propidium iodide exclusion (n>5 at each time point). The number ofday after death is shown on the x-axis and the number of cells labelledwith calcein is shown on the y-axis. This figure shows a lineardecreasing of cells alive in muscle tissue after 4 and 8 days afterdeath.

FIG. 2.B. illustrates the number of viable satellite cells (muscle stemcells) in a sample extracted from the tibialis anterior muscle ofTg:Pax7-nGFP transgenic mouse cadaver (n>5 at each time point). Thenumber of viable satellite cells was evaluated by Flow Cytometry on GFPexpression. The number of day after death is shown on the x-axis and thenumber of GFP positive cells is shown on the y-axis.

FIG. 2.C. illustrates the enrichment of Pax7 expressing satellite cellsextracted from the cadaver of knock-in mouse Pax7^(LacZ/+). Theenrichment was evaluated by measuring the percentage of X-Gal positivecells. The number of day after death is shown on the x-axis and thenumber of X-Gal+ cells is shown on the y-axis.

FIG. 2.D. illustrates the proportion of satellite cells that surviveafter death in a sample extracted from the cadaver of juvenileTg:Pax7-nGFP transgenic mouse. The juvenile mice in which all SCs areactivated have been used. This graph show the absence of decreasing ofSCs between day 0 and day 4 post mortem showing that activated SCs canalso resist 4 days post mortem. Eight days post mortem very few cellsremain alive. Results are expressed as number of cells per mg of tissue.The number of day after death is shown on the x-axis and the number ofGFP positive cells is shown on the y-axis.

FIG. 2.E. illustrates the viability of post mortem stem cells in aquiescent cell state Satellite cells were enumerated by FACS fromresting or injured TA muscle of Tg:Pax7-nGFP mice at day 0 or 4 dayspost mortem (pm).

FIG. 3.A. is a graph which illustrates the expression level of twosatellite cell key genes, i.e. Pax7 and MyoD, in skeletal muscle samplefrom mouse cadaver (n=5 at each time point). Gene expression wasassessed by real time PCR (Taqman). The number of day after death isshown on the x-axis and the expression of Pax7 and MyoD is shown on they-axis. 2-deltaCT indicates the threshold after which the signal isconsidered as significant and normalize with ubiquitously expressed gene(GAPDH: Glyceraldehyde 3-phosphate dehydrogenase).

FIG. 3.B. is a graph which illustrates the expression level of markersof myogenic cell commitment, i.e. Troponin, MyoD, Myogenin and, ofmarkers of stem-like state, i.e. Pax7 and CD34 in skeletal muscle samplefrom mouse cadaver (n=5 mice for each gene). Gene expression wasassessed by real time PCR (Taqman). The marker which is quantified isshown on the x-axis and the expression is shown on the y-axis. 2-deltaCTindicates the threshold after which the signal is considered assignificant and normalize with ubiquitously expressed gene (TBP: tatabinding protein)

FIG. 3.C. illustrates the percentage of clonogenicity of satellite cells(i.e. number of cells forming colonies) after FACS cell sorting andplating in 96well dishes at day0, 4 and 8 post mortem. The number of dayafter death is shown on the x-axis and the expression of the percentageclonogenicity is shown on the y-axis.

FIG. 3.D. illustrates the time culture which is necessary to achieve thefirst division as a function of the number of day following death. Thetime culture is shown on the y-axis and the number of day after death isshown on the x-axis. This graph shows that cells that remain alive 4 and8 days post mortem make their first division later than cells collectedimmediately after death.

FIG. 3.E. illustrates the measurement of the energizing state ofsatellite cells obtained from Tg:Pax7-nGFP transgenic mouse cadaver as afunction of the number of day following death. Comparison was performedbetween day0, day4 and day8 post mortem. The number of activemitochondria in the different conditions was assessed by Mitotracker.The relative intensity of fluorescence which is the result of thestaining of active mitochondria with Mitotracker is shown on the y-axis,and the ratio Day 0/Day X after death is shown on the x-axis.

FIG. 3.F. illustrates the quantity of ATP produced in satellite cellsobtained from Tg:Pax7-nGFP transgenic mouse cadaver as a function of thenumber of day following death. The quantity of ATP was determined byfluorescence using luciferase activity as a readbout. Bioluminescence isshown on the y-axis and the number of day after death is shown on thex-axis.

FIG. 4.A. illustrates the percentage of survival in lethally irradiatedmice which have been transplanted with bone marrow extracted from onesingle femur collected 0, 2, 3 and 4 days post mortem. The number of dayafter death is shown on the x-axis and percentage of mouse survival isshown on the y-axis.

FIG. 4.B. illustrates the percentage of blood chimerism (% of GFP+leukocytes in circulating blood) after direct transplantation (blackbars) and after a serial transplantation (gray bars) of bone marrow. Thenumber of days after death when bone marrow used for transplantation washarvested is indicated on the x-axis, and percentage of mouse survivalis shown on the y-axis. Blood chimerism after bone marrowtransplantation using cadaver BM is strictly identical to what observeat day 0 post mortem and decrease lightly when using BM from day 4 postmortem.

FIGS. 4.C-F. illustrate immunophenotyping of circulating GFP+ cellsafter serial bone marrow transplantation assessed by flow cytometry.

FIG. 4.C shows the percentage of GFP+ B cells (cells expressing B22Ocell surface marker).

FIG. 4.D shows the percentage GFP+ T cells (cells expressing CD5 cellsurface marker).

FIG. 4.E shows the percentage GFP+ granulocytes (cells expressing Gr1cell surface marker).

FIG. 4.F shows the percentage and GFP+ myeloid cells (cells expressingCD11b cell surface marker).

These experiments demonstrate the ability of cadaver bone marrow tofully reconstitute blood compartment.

FIG. 5. illustrates the number of cells that remain alive at 4° C. innormoxia (black bars) compared to complete anoxia (gray bars) as afunction of days.

FIG. 6.A. illustrates the percentage of whole bone marrow cell loss manydays after anoxia at 4° C. (black bars) Four days after storage inanoxia, 95.6% of cells are lost whereas bone marrow is stilltransplantable at this time point see FIG. 6B.

FIG. 6.B. illustrates the percentage of blood chimerism (% of GFP+leukocytes in circulating blood) after direct transplantation (blackbars) and after a serial transplantation (gray bars) of bone marrow.Transplanted was performed with bone marrow maintain 0 hours, 1 day, 2days, 3 days or 4 days in anoxia before being transplanted. The numberof day in which bone marrow was maintained in anoxia before beingtransplanted is indicated on the x-axis, and percentage of bloodchimerism is shown on the y-axis. Note that n>5 and that 100% of micesurvive at each time point.

FIGS. 6.C-F. illustrate immunophenotyping of circulating GFP+ cellsafter serial bone marrow transplantation assessed by flow cytometry.

FIG. 6.C shows the percentage of GFP+ B cells (cells expressing B22Ocell surface marker).

FIG. 6.D shows the percentage GFP+ T cells (cells expressing CD5 cellsurface marker).

FIG. 6.E shows the percentage GFP+ granulocytes (cells expressing Gr1cell surface marker).

FIG. 6.F shows the percentage and GFP+ myeloid cells (cells expressingCD11b cell surface marker).

EXAMPLE I Material and methods

Ethical

Human samples were collected according to guidelines recommended by thenational ethical committee.

All mice were housed in a level 2 biosafety animal facility, andreceived food and water ad libitum. Prior to manipulations, animals wereanaesthetized using intraperitoneal injection of Ketamine and Xylazine(respectively 25% and 12.5% in PBS). This study was conducted inaccordance with the local and EC guidelines for animal care (JournalOfficiel des Communautés Européennes, L358, Dec. 18, 1986).

Mouse Strain

C57BL/6 mice (Iffa-Credo, L'arbresle, France), Tg:Pax7-nGFP mice inwhich satellite cells can be easily visualize by their GFP expression(Sambasivan R, Dev Cell. 2009 June; 16(6):810-21), Pax7^(nlacZ/+) micein which satellite cells can be easily visualize by their LacZexpression (Ramkumar Sambasivan and Shahragim Tajbakhsh unpublished),Tg:Pax7-nGFP::Tg:CAG:PLAP (Sambasivan 2009) in which all cellsconstitutively express placental alkaline phosphatise and satellite cellGFP, Tg: CAG-GFP mice (C57BL/6 TgN[actEGFP]Osb YO1) in which the GFPtransgene is ubiquitinously expressed under the control of a non-tissuespecific promoter, chicken beta-actin with cytomegalovirus enhancer, asa cytoplasmic protein (Okabe, et al, FEBS Lett. 1997 May 5;407(3):313-9).

Tissue Preparation

Depending of the condition used, after animal sacrifice, tissues weresnap-frozen immediately in liquid nitrogen-cooled isopentane forimmunohistochemistry and histological analysis, or fixed using buffered4% paraformaldehyde prior to cryopreservation in 30% sucrose overnightat 4° C. (a procedure that keep the spontaneous fluorescence of GFP in atissue section). Serial 7 μm thick cryosections were performed foranalysis.

Images were captured on a Zeiss Axiophot microscope with an Apotome®(Carl Zeiss Inc., Germany) and Orca ER digital camera (HamamatsuPhotonics, Japan) using Simple PCI (C-Imaging, Compix Inc) software.

Immunohistochemistry

For human cases, 5 μm cryosections of muscles from cadavers wereimmunostained with mouse anti-human CD56 (1:20 dilution; NHK-1-RD1;Beckman Coulter) and revealed using peroxidase Vectastain ABC kit(Vector Laboratories).

For mouse tissues, immunostainings were done without antigen unmasking.

The following protocol was always used: after rehydratation of sectionswith PBS, non-specific protein binding was blocked with 20% goat serumand cells were permeabilised with 0.5% triton-X100 (Sigma-Aldrich,St-Louis, Mo.) 20 minutes. Incubation with primary antibody was doneovernight at 4° C., and signal was revealed with secondary antibodyincubated 1 hour at 37° C.

Dako Diluent buffer (Dako, Glostrup, Denmark) was used for dilutingantibodies.

The following antibodies were used as primary: mouse monoclonalantibodies against human Placental Alkaline Phosphatase [8B6](1:300,GenTex, Irvine, Calif.), M-cadherin (1:50, Alexis Biochemicals, Lausen,Switzerland), myogenin (1:50, BD Pharmingen, San Jose, Calif.), rabbitpolyclonal anti-human (or mouse) desmin (1:50, Abcam, Cambridge, UK),rabbit polyclonal antibodies against mouse myogenin (1:50, Santa Cruzbiotechnologies, Santa Cruz, Calif.) and Laminin-1 (1:50, Sigma-Aldrich,Saint-Louis, Mo.).

The secondary antibodies used was Cy3 conjugated donkey anti-mouse(1:400, Jackson Immunoresearch lab., Baltimore, Pa.), FITC conjugatedgoat anti-mouse (1:200, Jackson), Cy3 conjugated donkey anti-rabbit(1:200, Jackson), biotinilated horse anti-mouse (1:200, Vectorlaboratories, Burlingame, Calif.), Cy5 conjugated donkey anti-rabbit(1:200, Jackson). FITC conjugated donkey anti-goat (1:200, Jackson) andDTAF conjugated-streptavidin (1:400, Immunotech Beckman, Brea, Calif.).

X-Gal staining

Cytocentrifuged cells were rehydrated with PBS before fixation with PFA4% followed by overnight incubation with X-Gal 40 mg/ml (reconstitute inDiméthylsulfoxide, Invitrogen, Paisley, UK) in a solution containing 4mM each of potassium ferrocyanide, potassium ferricyanide, 2 mM MgCl,and 0.02% NP40 in PBS at 37° C.

Bone Marrow Transplantation

Briefly, donor BM cells were obtained by flushing 2 femurs of donor mice(various times post mortem) with RPMI medium (Invitrogen, Paisley, UK)and 0.1% heparin (Choay 5000 UI/ml). In the case of late post-mortemcadavers the BM cell suspension was incubated with 50 μg/ml DNAse I(Roche, Mannheim, Germany)I to preclude clogging of cells. Afterwashing, retro-orbital injection of cells was done in 0.1 ml fresh mouseserum and Hanks Buffer (PAA Laboratories GmbH, Pasching, Austria) (1:1),in 9.5 Gy-irradiated, 4 week-old B6 mice (⁶⁰Co γ rays within 1 daybefore BM transplantation). After transplantation, mice receivedmg/kg/day ciprofloxacin for 10 days to prevent infection during theaplastic phase.

Flow Cytometry Analysis

To quantify the amount of engraftment, the peripheral blood mononuclearcells of transplanted mice were analyzed by flow cytometry using a Cyan™cytometer (DakoCytomation, Glostrup, Denmark) 1 monthpost-transplantation. Red blood cells were lysed using “ACK” buffer(NH₄Cl 0.15M, KHCO₃ 1 mM, Na₂EDTA 0.1 mM) and immunostainings were doneat +4° C. for 30 nm using rat anti mouse CD16/CD32 (Mouse BD Fc Block™)(BD Biosciences) to preclude cell activation and adherence to plastic.In all the FC experiment cells were also labelled with Propidium Iodide1 mg/ml (Sigma-Aldrich, St-Louis, Mo.) to exclude dead cells fromanalysis.

Leukocytes were gated on, and GFP fluorescence was measured under thefluorescein isothiocyanate channel. Specific fluorescence stainings weredone using PE-Cy5-conjugated anti-Ly-6C (Gr1) (eBioscience San diego,USA). PE-conjugated anti-CD11b (eBioscience San diego, USA),PE-conjugated anti-CD5 (BD Biosciences), PE-conjugated anti-B220 (BDBiosciences), Abs and their respective isotypes. All analyses andquantitation were performed using Summit v4.3 software fromDakoCytomation.

For the assessment of active mitochondria immediately after isolation ofcells by FACS Mitotracker (invitrogen, M22246) deep red at 500 nM wasused for minutes at 37° C. Then the intensity of far red staining wasanalysed.

ATP Level Measurement

For measuring the levels of ATP, cells were isolated by FACS directly inlysis buffer and maintained at 4° C. An ApoSENSOR™ kit from biovision(Catalog #K254-200, -1000) was used to measure ATP levels whereluciferin reacts with ATP and emits signal in proportion to ATP content;emitted light was measured using luminometer (GLOMAX 20/20 luminometerpromega).

FACS Cell Sorting and Analysis

MoFlo Legacy (Beckman Coulter, Brea, Calif.) was used for cell sortingand CyAn ADP for cell analysis (Beckman Coulter).

Cell Suspension Preparation from Muscle Tissue

After sacrifice, muscles from mice were carefully dissected, minced insmall pieces and washed in PBS before digestion with Pronase (proteasefrom streptomyces griseus (Sigma-Aldrich, St-Louis, Mo.) reconstitutedin DMEM with penicillin Streptomycin 0.4%). All supernatants werecollected and enzyme activity immediately blocked by adding 20% foetalcalf serum. This procedure was performed serially until completedigestion of the tissue (4 to 5 rounds of 20 minutes digestion at 37°C.). Cells were then washed and filtered with a 40 μm cell strainerbefore 10 minutes treatment with an antibiotic/antifungus cocktail.

Cell Cultures

Unless otherwise indicated, culture media components were obtained fromGIBCO (Invitrogen, Paisley, UK) and culture plastics were obtained fromTPP (Trasadingen, Switzerland). Human or mouse muscle cells werecultured from muscle samples as described previously (Chazaud et al.,2000). In standard conditions (spontaneous in vitro myogenesis), cellswere grown in Ham's F12 medium containing 20% FCS (growing medium) 1%UltroserG (PALL Life Sciences, Saint Germain en Laye, France), 0.2%Vitamins, 1% non essentials amino acids 100X, 0.4% PenicillinStreptomycin 10000 U/ml without serum withdrawal. In differentiatingconditions, growing medium was replaced by Ham's F12 medium containing5% FCS (differentiating medium) at time of subconfluence.

For culture without oxygen, GenBag® (Biomerieux, Craponne, France)devices were used.

RNA Extraction, RT and qPCR

Total RNA was extracted from cells isolated by FACS on GFP positivitydirectly in lysis buffer using the Quiagen RNAeasy Micro purificationKit. 400-600 ng of DNAse-treated (Roche). RNA was processed forrandom-primed reverse transcripion using the SuperScript II reversetranscriptase protocol of Invitrogen. The cDNAs were then analyzed byreal-time PCR using Taqman universal Master Mix and an ABI Prism 7700(Perkin-Elmer Applied Biosystems) and a StepOnePlus (AppliedBiosystems). TBP reference transcript levels were used for thenormalisation of each target within each sample (=ΔCT). Custom primerswere designed using the Primer3Plus online software.

Statistical Analysis

In all experiments the “n” value was at least 5. The t test was used forstatistical analyses (GraphPad-InStat® software). P<0.05 was consideredsignificant.

EXAMPLE II Stem Cells Survive for Extended Periods Post Mortem

To determine how long muscle cells would survive in dead tissue, humancadavers were obtained from the “centre du don des corps—Faculté deM-décine Paris Descartes”. After death, cadavers were store at 4° afteran initial and variable period lasting from several hours to 24 hours atroom temperature. In all cases (n=16) patients were from 57-95 y.o. inage (mean 84 y.o.). A deltoid muscle biopsy (2 grams) was performed from6-17 days post mortem. None of the patients were suffering fromneoplasia. Histological analysis of the muscle showed a necroticappearance and chromatin from myonuclei usually appeared leaky. CD56immunostaining which labelled satellite cells (SCs) (i.e. muscle temcells) in human showed a few positive cells were not necrotic, but theyexhibited a compact appearance.

Mononuclear cells were extracted from muscle biopsies using standardprotocols (Chazaud B, et al. Exp Cell Res. 2000; 258: 237-44.) andcultured for two weeks in gelatin coated dishes and in a “classical”medium composed with HamF12, 20% fetal calf serum, 0.4%penicillin/streptomycin, 1% ultroserG®, 0.2% vitamin, 1% non essentialamino-acids. In all cases, including latepost mortem time point (17days), after a maximum of 4 days, a few cells were observed that wereattached to the bottom of the dish. They grew slowly from small coloniesand when the density reached a critical threshold, some cells align tofuse. Two weeks post plating, differentiating medium (HamF12, 5% normalhorse serum, 0.4% penicillin/streptomycin, 0.2% vitamin, 1% nonessential amino-acids) was added to the culture and cell fused formingnumerous myotubes.

Immunostainings confirm that more than 90% of the attached cells formingsmall colonies were expressing the myogenic marker Desmin. This was alsothe case at later stages when these cells fused and differentiated intomyotubes, expressing both Desmin and the differentiation transcriptionfactor Myogenin. Due to the extensive decomposition of tissues, we werenot able to obtain cadavers after 17 days post mortem.

To test the survival potential of SCs in muscle samples, we takeadvantage of organ donors with beating heart in who we perform asurgical muscle biopsy. These Donors were younger in age (n=15; from41-77 y.o., mean 57 y.o.). We kept the muscle sample in a bufferedmedium (DMEM, 1 mM HEPES, 0.4% penicillin/streptomycin), at 4° C. in asealed container. The time of tissue sampling (i.e. number of daysbetween the sampling and the culture) was noted (see Table I below).

TABLE I D2 D4 D6 D10 D14 D20 D25 D30 D35 D40 D50 D55 D60 D77 F 56y.o. + + + F 58 y.o. + + − − − F 43 y.o. + + − − − M 50 y.o. − − F 59y.o. + + − F 57 y.o. + + + + − − F 74 y.o. + − M 69 y.o. + + + + + M 55y.o. − − F 43 y.o. + + + − − M 65 y.o. + + − − M 54 y.o. − M 55 y.o. + +− F 41 y.o − F 77 y.o + + −

As observed in cadavers, and from day 4 post-biopsy, muscle exhibited anecrotic appearance with some remaining CD56 immunopositive and othercompact small cells adjacent to myofibers. Depending of the size of thesample, culturing muscle cells was possible many days after sampling.The samples were assayed regularly from day 2-77. Prior to day 30post-sampling, the cultured cells yielded large numbers of cells, themajority (>80%) being myogenic as assessed by the formation of myotubesthat expressed Myogenin and Desmin. After 35 days post-sampling viablecells were no longer obtained (assayed for 15 days in culture).

Similar results were obtained with mouse cadavers. C57BL6 mice (n=10 pertime point) were sacrificed using CO₂ and kept for several days at 4° C.(FIG. 1). Skeletal muscles displayed a necrotic and oedematousappearance with some remaining M-cadherin expressing cells a marker forSCs. Muscle SCs were cultured up to 10 days after isolation. One hundredpercent (n=10) of the cases gave rise to a large number of SCs. After 10days post mortem, most of the cases did not yield viable cells inculture, in part because of the contamination of the medium by bacteriaarising during tissue decomposition. Mouse cadavers were more sensitiveto bacterial proliferation (even at 4° C.) than humans. Up to ten dayspost mortem, all cultured cells were myogenic as assessed by theformation of myotubes and the expression of myogenic markers.

EXAMPLE III Characterisation of Cell Types that Survive after OrganismalDeath

To determine if stem cells have a greater capacity to survive afterorganismal death, cell suspensions obtained from cadavers were stainedwith calcein that labels only live cells and evaluated the number ofcells that remained alive by flow cytometry (FC). As shown in FIG. 2.A.the number of cells incorporating calcein per mg of tissue (n=7 animalsat each time point) varies from 2678±718 at day 0 to 820±33 at day 4 and179±22 at day 8 post mortem.

To determine the number of viable SCs in a tissue after organism death,the Tg:Pax7nGFP mice were used in order to take advantage that all theirsatellite cells are GFP-positive and that SCs could be prospectivelyisolated by FACS based on GFP epifluorescence (Sambasivan R, Dev Cell.2009 June; 16(6):810-21). The number of SCs in one Tibialis anterior(TA) muscle was enumerated every four days after death from 8 week oldmice kept at 40° C. As shown in FIG. 2.B. four days after death, 50% ofSCs remained in the TA. Eight days after death, 30% of the SCs survivedin the TA. By 12 or 16 days post mortem only a few viable SCs remained(2% and 1%, respectively). All GFP+ cells isolated by FACS were viableas assayed by the exclusion of propidium iodide, and the ability to givecolonies when cultured.

To determine the proportion of SCs that survive after death, theknock-in mouse Pax7^(nlacZ/+) was used. In this mouse, bacterial lacZreporter gene expression reflects the expression of the Pax7 gene in allsatellite cells (R. Sambasian and S. Tajbakhsh, unpublished). Betweenday 0 and day 4 post mortem, the proportion of X-gal positive cellsincreased by 3.4 fold (see FIG. 2.C.) indicating that a higherproportion of muscle stem cells were present after organismal death.

To investigate the mechanism that permits muscle stem cells to surviveafter organismal death, it was examined whether cellular quiescenceconferred a survival advantage compared to proliferating cells. To dothis, we counted SCs from Tg:Pax7-nGFP in juvenile mice before SCsentered quiescence (growth paradigm). At P10 (10 days postnatal),satellite cells proliferate actively and myofibres continue to increasein size due to the addition of nascent myoblasts (Shinin V, et al. NatCell Biol. 2006 July; 8(7):677-687; White R B, et al. BMC Dev Biol. 201022; 10:21). In this scenario, no significant drop in SCs number betweenday 0 and day 4 post mortem (FIG. 2.D.) was observed after sacrifice ofthe pups. However, dramatic decrease was observer after 8 days postmortem.

To confirm that quiescent state might confer a survival advantage to thestem cells, three cohorts of mice were examined. In the first, satellitecells were enumerated from uninjured TA muscle of Tg:Pax7-nGFP mice at 4days post mortem (1980±212 GFP⁺/TA; n=5 mice; see FIG. 2.E). Satellitecells in the second cohort were enumerated 5 days after a severe muscleinjury with a myotoxin, thereby promoting stem cell re-entry into thecell cycle to effect myofibre regeneration. At this time, myogenic cellsproliferate actively (mean 103,000±8494 GFP⁺/TA; n=5 mice; see FIG.2.E). The third cohort was treated similarly, but 5 days post-injury,mice were sacrificed, then satellite cells were isolated by FACS at 4days post mortem (mean 460±80 GFP⁺/TA, n=5 mice). Therefore, themajority of proliferating myogenic cells do not survive in post mortemtissue suggesting that cellular quiescence, in part, protects stem cellsfrom death in post mortem tissue.

EXAMPLE IV Characterisation of Viable Cells after Organismal Death

To characterise the surviving SCs sub-population after death in mouseskeletal muscle, RT-qPCR were performed on purified satellite cellsisolated by FACS and lysed directly in buffer. A library of cDNA wassynthesized by reverse transcription and real time PCR (Taqman) wasperformed to assess the gene expression level of key satellite cellgenes. The level of Pax7 and MyoD were similar in surviving cells day 4and day 8 post mortem vs. cells extracted immediately after death (n=5)(FIG. 3.A.). Complementary experiments were carried out to furtherassess the extent of lineage priming and hence, the commitment status ofthe muscle stem cells post mortem. To do that, we performed RT-qPCR onpurified satellite cells isolated by FACS. Cell determination anddifferentiation markers Myod and Myogenin, and the myofibre structuralprotein Troponin T were used as readouts for myogenic cell commitmentwhereas Pax7 and the receptor stem cell marker CD34 were used asreadouts of the more stem-like state. Interestingly, a progressiveincrease in the levels of CD34 was observed from day 0 to day 8 postmortem, whereas an inverse trend was noted for MyoD, Myogenin andTroponin T transcript levels (n=5 mice/condition; see FIG. 3.B). Thissuggests that the post mortem derived muscle stem cells are lesstranscriptionally primed for myogenic commitment compared to thoseisolated from freshly isolated tissue.

Further, these data clearly show that the post mortem derived musclestem cells are characterized by a lack of detectable expression ofMyogenin gene, while muscle stem cells extracted immediately after death(which is considered as representing cells present in a living subject)do express myogenin.

To assess the functional potential of surviving satellite cells, clonalanalysis of cells sorted from Tg:Pax7-nGFP mice was performed. Thepercentage of clonogenicity (i.e. percentage of cell forming coloniesafter FACS in a 96 well plate) was not significantly different betweenday 0 and day 4 post mortem (20% vs. 16.3%) but this value dropsdramatically at day 8 post mortem (1.6%) (FIG. 3.C.). This suggestsstrongly that satellite cells are able to resist to stress from theenvironment after organismal death. All colonies were myogenic asassessed by contracting myotube formation after differentiation.Although potential to form colonies was equivalent between day 0 and day4 post mortem, we observed that exit from cell quiescence, assessed byscoring the first division after plating, was longer in post mortem (29hours) sorted cells in comparison to alive animals (21 hours) (see FIG.3.D.). After the first division cells divided every 7 hours andsynchronously in the tested culture conditions in both situationsindicating that cells recovered their correct cell cycle time afterculture from dead animals.

To characterize further the sub-population of resisting cells in ahostile environment, their energizing state was measured by assessingthe mitochondrial number and activity, as well as ATP level in SCs 4days post mortem. To do this, SCs were isolated by FACS fromTg:Pax7-nGFP mice from day 0, day 4 and day 8 animals post mortem.Staining with Mitotracker allowed assessing the number of activemitochondria in the different conditions by flow cytometry. The numberof active mitochondria was significantly diminished in post mortemsamples compared to live control adult animals. Interestingly, as shownin FIG. 3.E., no significant difference in this value was observedbetween day 4 and 8 post mortem. The level of ATP in the 3 conditionswas also monitored (alive —day 0—, day 4 and 8 post mortem). Cells wereisolated as indicated above. For all 3 conditions, 20,000 cells wereused and this number was double-checked by direct counting with aMalassez Chamber®. The quantity of ATP was determined by fluorescenceusing luciferase activity as readout. Like mitochondrial activity, thequantity of ATP dropped dramatically in day 4 and day 8 post mortem incomparison to the day 0 “alive” controls (FIG. 3.F.).

Taken together these results reveal a direct correlation between theenergizing threshold of the cell, presumably to ensure essential basalcellular activity and maintain viablilty, and the capacity to resist toa hostile environment. Cells exhibiting values below this threshold arenot viable. These readouts provide insights into the mechanisms thatallow these stem cells to survive after organismal death, and theyprovide a powerful tool to be used in diagnostic and therapeuticalpurposes.

To determine if stem cells have the functional capacity to regenerate atissue after transplantation, SCs were extracted 4 days post mortem andengrafted into preinjured regenerating skeletal muscles ofimmunocompromised Rag1/2^(−/−): C^(−/−) recipient mice. Donor mice weredouble transgenic Tg:Pax7-nGFP::Tg:CAG-PLAP (PLAP, human alkalinephosphatase) mice to prospectively isolate satellite cells by FACS usingGFP. The ubiquitous reporter PLAP permits to follow the fate of theengrafted cells.

In all the cases a significant contribution to regenerating skeletalmuscle by the donor population was observed. After engrafting 10,000 SCsextracted from day 4 post-mortem mice, a mean of 300 PLAP-expressingmyofibers were obtained. This result is similar to what is observedusing control freshly isolated satellite cells.

EXAMPLE V Assessment of the Viability and Engraftment Potential ofHaematopoietic Stem Cells Isolated from Mice Post Mortem

To determine if this extreme resistance to post mortem conditions isonly the case for skeletal muscle stem cells, or another stem cellpopulation behaves in a similar manner, hematopoietic stem cells werestudied. At daily intervals post mortem, the bone marrow (BM) ofTg:CAG-GFP mouse femur was flushed (two limbs) and kept at 4° C. BMtransplantations were performed in lethally irradiated C57BL6 recipientmice. The engraftment potential of transplanted BM progenitors wasassessed by the percentage of GFP+ leukocytes found in the circulatingblood. Using, 2, 3 and 4 days post mortem BM, blood cells were readilyand fully reconstituted by BM progenitors in lethally irradiatedrecipients (n>5 in each case). Viability was ensured with all theanimals that received a BM transplantation except when using BM from 4days post mortem where viability was 60% (FIG. 4.A.). In all theseanimals GFP+ cells represented more than 70% of leukocytes, a resultnormally found in controls (donors) corresponding to 100% chimerism(FIG. 4.B.) After serial transplantation, same results were obtainedexcept at day 4 where only 50% chimerism was found. In all theseexperiments 100% of animals survive. In all the cases, GFP+ leukocyteswere found in all lineages: lymphocytes B, lymphocytes T, granulocytes,or monocytes as assessed by flow cytometry using B220, CD5, Gr1 or CD11bexpression, respectively (FIGS. 4.C. to 4.F.).

To determine if the cells that were extracted from post mortem BMcontained long term hematopoietic stem cells, a serial transplantationwas performed with the grafted BM. GFP+ cells from the bone marrow ofpreviously grafted animals were isolated by FACS 2 months after thefirst transplantation and re-grafted in lethally irradiated recipients.In all the cases, independently of the source of the initial BM (day 2,3 or 4 post mortem), all of the animals were viable with 100% chimerism,and we obtained GFP+ leukocytes in all the different lineages.

EXAMPLE VI Anoxia and Low Temperature Enhance the Viability andTransplantation Potential of Stem Cells Isolated Post Mortem

To investigate the mechanism which confers the observed resistance ofstem cells, the hostile environment occurring after death in a tissuei.e. hypoxia followed by anoxia was modeled. Culture conditions wasestablished with a device usually used to culturing anaerobic bacteria(GenBag Chambers®). The cells were maintained at 40 for various timeintervals in the absence oxygen (less than 0.1% according tomanufacturer). SCs isolated by FACS from Tg:Pax7-nGFP mice weremaintained at 4° C. for 4, 7, 14 and 21 days in the absence oxygen (lessthan 0.1% according to manufacturer). Strikingly, it was observed thatSCs better survived in a complete anoxic environment than in normoxic(20%) environment at 4° C. (see FIG. 5.A.). As an example, after 4 daysat 4° C., 82.3% of SCs were lost in the normoxia condition compared to28.9% in anoxia and after 7 days at 400, 99.7% of cells were lost innormoxia compared to 97.7% in anoxia (FIG. 5.). The functional capacityof these cells was maintained after several days without oxygen asassessed by their ability to grow and differentiate when cultured undernormal conditions. To test the functional capacity of these cells ingreater detail, SCs from Tg:Pax7-nGFP::Tg:CAG-PLAP donor mice wereisolated by FACS and maintained at 4° C. with or without oxygen until 4days days. After this period, the cells were transplanted them byintramuscular injection into pre-injured TA muscles of C57BL6 recipientmice. These results demonstrate that cells maintained without oxygen hadat least the same transplantation capacity than those maintained withoxygen.

Similar experiments were performed with hematopoietic stem cellsisolated from mice post mortem and stored in the presence or absence ofoxygen. BM cells were extracted from two femurs of Tg:CAG-GFP animals,kept at 4° C. for 1, 2, 3, or 4 days, and transplanted into lethallyirradiated C57BL6 recipient mice (n≧5). Cell mortality in suchconditions was important i.e. 63+/−7% of cell loss compare toimmediately after extraction after one day in anoxia, 79+/−3% after twodays in anoxia, 98+/−1 after 3 days in anoxia and 96+/−1 after 4 days inanoxia (FIG. 6A). Even with this important cell loss, number of cellswas sufficient enough to graft into irradiated recipients and bloodchimerism was slightly 100% (FIG. 6.B. black bars) and all hematopoieticlineages were reconstituted (FIGS. 6.C. to 6.F.). Serialtransplantations of BM cells isolated after the first round oftransplantation demonstrated that the HSCs had a long term capacityrepopulate the lineage (FIG. 6.B. grey bars).

In summary, the inventors have shown in animal model thattransplantation of skeletal muscle and hematopoietic stem cells obtainedfrom cadavers by the method of the invention, or enriched frombiological sample when maintained in the absence of oxygen arefunctional and contribute to the regeneration of their respectivetissues.

1. A method for obtaining mammalian stem cells comprising the followingsteps: a) harvesting a tissue from a mammalian cadaver stored at 1-6°C., the harvesting being performed on a part of the cadaver's body inwhich stem cells are usually present in a living counterpart; and b)extracting mononuclear cells from the harvested tissue.
 2. The methodaccording to claim 1, wherein the method comprises an additional stepb′) following step b) consisting of culturing the extracted mononuclearcells.
 3. A method for obtaining mammalian stem cells expressing atransgene of interest, comprising the following steps: a) harvesting atissue from a mammalian cadaver stored at 1-6° C., the harvesting beingperformed on a part of the cadaver's body in which stem cells areusually present in a living counterpart; and b) extracting mononuclearcells from the harvested tissue; b′) optionally culturing the extractedmononuclear cells of step b); and c) transforming or transfecting with avector, especially a vector of expression, or transducing with a virusvector, the mononuclear cells of step b), or step b′) where appropriate,said vector comprising at least one polynucleotide sequence of interest.4. The method according to claim 1, wherein the mammalian cadaver wasstored at 1-6° C. from 0 minute to 48 hours after death.
 5. The methodaccording to claim 1, wherein step a) is performed after a period oftime following death comprised between 0 minute and 30 days.
 6. Themethod according to claim 1, wherein the mammalian cadaver is a humancadaver.
 7. The method according to claim 1, wherein: step a) isperformed on trabecular bone or peripheral blood; the harvested tissueis bone marrow or peripheral blood; and the stems cells obtained arehematopoietic stem cells, peripheral hematopoietic stem cells ormesenchymal stem cells.
 8. The method according to claim 1, wherein:harvesting of step a) is performed on muscle, especially skeletal orsmooth muscle; the harvested tissue is muscle sample, especiallyskeletal or smooth muscle sample; and the stems cells obtained aremuscle stem cells.
 9. The method according to claim 1, wherein:harvesting of step a) is performed on brain or spinal cord at least 24hours after death; the harvested tissue is brain, spinal cord ormeninges sample; and the stems cells obtained are neural stem cells 10.The method according to claim 1, further comprising an ultimate stepfollowing step b), step b′) or step c) where appropriate, consisting ofresuspending the extracted or cultivated mononuclear cells in apharmaceutically acceptable carrier.
 11. A method for obtaining stemcells comprising the following steps: a) maintaining a biologicalmaterial which usually comprises stem cells at an oxygen concentrationequal to or less than 0.1%; and b) selecting viable cell, wherein viablecells are stem cells.
 12. A method for obtaining mammalian stem cellsexpressing a transgene of interest, comprising the following steps: a)maintaining a biological material which usually comprises stem cells atan oxygen concentration equal to or less than 0.1%; and b) selectingviable cell, wherein viable cells are stem cells. c) transforming ortransfecting with a vector, especially a vector of expression, ortransducing with a virus vector, the viable cells of step b), saidvector comprising at least one polynucleotide sequence of interest. 13.The method according to claim 11, wherein the biological material isobtained from a living donor, except human embryo or foetus.
 14. Themethod according to claim 11, wherein the biological material ismaintained at 1-37° C. during a period of time of 12 hours to 60 days.15. The method according to claim 11, wherein the biological material ismaintained at 1-37° C. in the absence of oxygen.
 16. The methodaccording claim 11, wherein the biological material is selected from thegroup consisting of bone marrow and circulating or peripheral blood, andwherein the selected viable stem cells are hematopoietic stem cells,circulating or peripheral hematopoietic stem cells or mesenchymal stemcells.
 17. The method according to claim 11, wherein the biologicalmaterial is muscle sample, especially skeletal or smooth muscle sample,and wherein the selected viable stem cells are muscle stem cells. 18.The method according to claim 11, wherein the biological material isselected from the group consisting of brain, spinal cord and meningessample, and wherein the selected viable stem cells are neural stemcells.
 19. The method according claim 11, wherein the biologicalmaterial was obtained from a mammal cadaver stored at 1-6° C. from 0minutes to 48 hours after death.
 20. The method according to claim 11,wherein the biological material was obtained from a living mammal. 21.The method according to claim 11, further comprising an ultimate stepfollowing step b), or step c) where appropriate, consisting ofresuspending the extracted or cultivated mononuclear cells in apharmaceutically acceptable carrier.
 22. Stem cells obtained accordingto the method of claim 11, for use in the regeneration of an injuredtissue.
 23. A method for culturing mammalian stem cells comprising usingan anaerobic cell culture system for culturing mammalian stem cells atan oxygen concentration equal to or less than 0.1%.
 24. The methodaccording to claim 23, wherein mammalian stem cells are cultured at1-37° C. for 12 hours to 30 days.
 25. The method according to claim 24,wherein the mammalian stem cells are Hematopoietic stem cells or musclestem cells.
 26. A method for selecting stem cells comprising using ananaerobic cell culture system for selecting for stem cells bymaintaining at an oxygen concentration equal to or less than 0.1% abiological sample comprising stem cells and non-stem cells.
 27. Anisolated muscle stem cell characterized in that it does not expressmyogenin gene (Myogenin−).
 28. An isolated muscle stem cell according toclaim 27 characterized in that it is Pax7high and/or of CD34+.
 29. Anisolated muscle stem cell according to claim 27, characterized in thatit has a low level of mitochondrial activity.
 30. An isolated musclestem cell as defined in claim 27, for use in the regeneration of aninjured tissue.